JPH0740948B2 - Microbiological production of amides - Google Patents

Abstract

A method of producing an amide comprises subjecting a nitrile to the action of a microorganism belonging to the genus Rhodococcus and having the ability to hydrate the nitrile, and isolating the resulting amide. The micro-organism is highly active to a wide variety of nitrile even at low temperatures and, in addition, the hydration reaction of nitriles has a high selectivity for the corresponding amides. Accordingly, this method ensures the production of amides in high yield without formation of by-products such as corresponding carboxylic acids.

Description

DETAILED DESCRIPTION OF THE INVENTION [3-1 Industrial Field of Application] The present invention relates to a method for producing a microbe of amide. More specifically, the present invention provides that the nitrile is treated with Rhodococ
The present invention relates to a microbiological method for producing an amide by subjecting it to a hydration reaction by the action of a specific microorganism belonging to the genus cus) and separating and obtaining the produced amide. Acrylamide and methacrylamide are known as industrially important amides.
Acrylamide has already been used as a polymer flocculant, a paper strengthening agent, a fiber improving agent, and the like, and a new application for the tertiary recovery of petroleum is being opened. On the other hand, methacrylamide is used for paints, adhesives, photo-crosslinkable compounds, etc. by taking advantage of its properties such as appropriate hydrophilicity, lipophilicity, excellent heat resistance and crosslinkability. The microbiological production method of the present invention can be used to efficiently and industrially produce these useful amides.

[3-2 Prior Art] As a method for producing an amide, a method in which nitrile is produced by hydrating copper in a reduced state as a catalyst is known, but it is difficult to regenerate the catalyst, and the amide produced is separated. The purification process is complicated, and further, the development of a new industrially advantageous manufacturing method has been desired.

On the other hand, recently, some methods for producing amides from nitriles using microorganisms have been proposed. For example Bacillus
llus genus, Bacteridium genus, Micrococcus genus, Brevibacterium (B
revibacterium) (JP-A-51-86186),
Corynebacterium (Corynebacterium) genus, a method using Nocardia (Nocardia) genus (Japanese Patent Publication No. 56-17918),
A method using the genus Pseudomonas (Japanese Patent Publication No. 59-37951) and the like.

[3-3 Problems to be Solved by the Invention] However, any conventional method using Bacillus genus, Bacterium genus, Micrococcus genus, Brevibacterium genus, Corynebacterium genus, Nocardia genus, Pseudomonas genus, etc. However, the amidation reaction activity is low, and the amount of organic acid by-produced in most of the conventional methods has not reached a level that is industrially sufficiently satisfactory.

[3-4 Means for Solving Problems] The inventors of the present invention hydrated nitrile and searched for a microorganism having an amidation reaction activity and selectivity that are industrially sufficiently satisfied, and investigated the culture and reaction conditions. As a result of intensive research, a microorganism having a high amide-producing ability was discovered among the microorganisms belonging to the genus Rhodococcus, and the present invention was completed. That is, according to the present invention, the raw material nitrile is subjected to a hydration reaction by the action of a microorganism to be converted into a corresponding amide, and the amide produced is separated from the reaction mixture, in which the microorganism is Rhodococcus (Rhodococcus). ) A microbiological process for the production of amides is provided which is characterized by the use of a specific microorganism belonging to the genus and having the ability to hydrate nitrites and the use of derived nitriles in the cultivation of said microorganism.

The microorganism used in the present invention is a specific amide-producing bacterium belonging to the genus Rhodococcus, and specifically, Rhodococcus
sp. AK32 strain (R.SP.AK32) (hereinafter abbreviated as AK32) and Rhodococcus sp. AK33 strain (R.sp.AK33) (hereinafter abbreviated as AK33). In addition, the Rhodococcus erythropolis AK3132 strain (R. erythropol) also found by the present inventors
is AK3132) (hereinafter abbreviated as AK3132) is also useful, but as will be apparent from the reference examples described later, since the yield of amide is low, it is described in the present invention for reference. AK3
2, AK33 and AK3132 are the Micromachine Research Institute No. 1046, 10
Deposited as No. 47 and No. 1040 at the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology.

The bacteriological properties and chemotaxonomic properties thereof are shown below.

The above-mentioned mycological and chemotaxonomic properties are described in the Bergy's Manual of Determinative Bacte
riology) 8th Edition (1974) and The Procaliotes (The
Prokaryotes, A Handbook on Habitats, Isolation, and
Classification based on Identification of Bacteria (1981), AK32, AK33 and AK3132 are aerobic, Gram-positive,
It is a non-motile rod-shaped bacterium, which is initially long rod-shaped but does not show hyphae, shows blanching and fragmentation, and later breaks into spheres or short rods. In addition, cell wall peptidoglycan has meso-diaminopimelic acid,
The N-acyl group is of glycolyl type. In the cell fatty acid analysis, it is mainly composed of straight-chain fatty acids and characteristically has 10-methyl-octadecanoic acid with the presence of mycolic acid. Therefore, it was determined that AK32, AK33 and AK3132 are bacteria belonging to the genus Rhodococcus. Differences between AK32, AK33, and AK3132 are recognized in terms of growth conditions and production of acid from sugar. These microorganisms have been deposited with the following numbers at the Institute of Microbial Technology, Institute of Industrial Science and Technology.

Next, general embodiments of the present invention will be described. Examples of the raw material nitrile subjected to the hydration reaction in the present invention include, for example, acetonitrile, propionitrile, succinonitrile, aliphatic saturated nitriles such as adiponitrile, acrylonitrile, and aliphatic unsaturated nitriles such as methacrylonitrile, Aromatic nitriles such as benzonitrile, phthalodinitrile and heterocyclic nitriles such as nicotinonitrile.

For culturing the microorganism used in the present invention, a derivative nitrile (inducer nitrile) selected from propionitrile, isobutyronitrile and methacrylonitrile (indutor nitrile) (indu
cer nitrile) as the only carbon source or nitrogen source, or a carbon source such as glucose or aldose, a nitrogen source such as ammonium sulfate or ammonium nitrate,
In organic nutrient sources such as yeast extract, malt extract, peptone, and meat extract, coexistence of derivatized nitrile as a carbon source and a nitrogen source, inorganic substances such as phosphate, sodium, potassium, iron, magnesium, manganese, and zinc. By culturing using a medium that appropriately contains a nutrient source and the like, cells having the hydration activity of the raw material nitrile can be obtained. The amount of the derivatized nitrile used as an inducer in the culture of the microorganism is not particularly critical, and may be an amount sufficient to give an amide-forming activity to the microorganism in the nitrile hydration reaction using the microorganism. The amount of the derivatized nitrile used in the culture of the bacterium during the amide-forming reaction of the nitrile using other existing bacteria is about the same. Of medium
pH is usually 5-9, preferably pH 6-8, temperature is usually 20-3
The culture is performed aerobically at 5 ° C, preferably 27 to 32 ° C for 2 to 5 days. In the present invention, "to convert a nitrile into a corresponding amide by subjecting it to a hydration reaction by the action of a microorganism" means that the microorganism is cultured in the presence of the nitrile to be hydrated, and the cell culture after the microorganism culture. When a substance, a microbial cell collected from the microbial cell or a treated product of the microbial cell, for example, a crushed product of the microbial cell, and the raw material nitrile are brought into contact with each other, both of them are included. It also includes the case where the cells are immobilized and used in the reaction. Collection of bacterial cells from the bacterial cell culture can be performed by a known method such as a centrifugation method,
The disruption of the cells can be performed mechanically with a homogenizer or the like, or can be performed using ultrasonic waves or the like.

In the present invention, as described above, the bacterial cell culture can be used as it is. Alternatively, if the bacterial cells and the treated bacterial cells separated by the above method are suspended in water or a buffer solution such as a phosphate buffer (for example, pH 7 to 9), and a raw material nitrile is allowed to coexist with the suspension, the hydrate is rapidly hydrated. The reaction proceeds and amide is produced. That is, usually, an aqueous suspension containing the microbial cells or a treated product of the microbial cells, for example, 0.1 to 5% by weight, and a raw material nitrile, for example, 0.2 to 10% by weight,
The reaction time may be, for example, 2 minutes to 8 hours under the conditions of temperature of 0 to 30 ° C. and pH of 5 to 10, for example. Further, since the raw material nitrile used as a substrate in the reaction is generally highly biotoxic, the concentration of the substrate in the reaction system is controlled to a level at which the reaction is not inhibited, for example, 2% by weight or less, and the microorganisms are added successively to the microorganism. Deactivation can be suppressed. Also, lowering the reaction temperature is effective in prolonging the life of microorganisms.

Thus, the starting nitrile can be converted to the amide in almost 100% molar yield without the formation of by-product carboxylic acid. The amide product thus obtained is purified according to a purposeful known method to obtain an amide product. That is, for example, from the reaction solution as it is or after removing the bacterial cells by centrifugation, membrane separation, etc.,
If necessary, remove colored substances, impurities, etc. by treatment with activated carbon, ion exchange resin, etc., then perform membrane separation,
The desired amide product can be obtained by concentration under reduced pressure, crystallization, centrifugation and the like.

[3-5 Effects of the Invention] The present invention uses a microorganism belonging to the genus Rhodococcus having hydration activity of nitrile, and thus has extremely high reaction activity when producing a corresponding amide from a raw material nitrile, and further has selectivity. It provides an industrially excellent method for producing an amide, which has almost 100% of the by-product and very little by-product.

In general, lowering the reaction temperature extends the life of microorganisms but decreases the reaction rate. In the method of the present invention, since a microorganism having extremely high reaction activity is used, when a predetermined reaction rate is obtained, the reaction temperature can be lowered, and as a result, the microbial life can be lengthened and the amide production cost can be reduced. I can.

[3-6 Examples] Next, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the Examples.

Example 1 (1) Culture AK32 was cultured under the following conditions.

1) Medium Tryptone 1.7 wt% Soypeptone 0.3 〃 Glucose 0.25 〃 Isobutyronitrile (inducer) 0.5 〃 Salt 0.5 〃 Dipotassium Phosphate 0.25 〃 pH 7.3 2) Culture conditions 30 ℃ / 4 days (2) Methacrylonitrile Hydration reaction of the bacterial cells was collected from the obtained culture solution by centrifugation, washed with physiological saline and used for the reaction. That is,
A reaction solution containing 0.2% by weight of dry cells, 2.0% by weight of methacrylonitrile and 97.8% by weight of 0.05M phosphate buffer solution (pH 7.0) was prepared, and the reaction was started at 30 ° C. Reaction start 5
After a minute, the reaction solution was analyzed by gas chromatography, and it contained 2.5% by weight of methacrylamide and no unreacted methacrylonitrile, methacrylic acid and other by-products, and the reaction proceeded almost quantitatively and was completed. Was.

Comparative Example 1 (1) Culture Corynebacterium sp.
The N-774 strain (Microtechnology Research Institute No. 4446) was cultured under the following conditions.

1) Glucose 1.0% by weight Peptone 0.5 〃 Yeast extract 0.3 〃 Malt extract 0.3 〃 pH 7.2 2) Culture conditions 28 ℃ / 3 days (2) Hydration reaction of methacrylonitrile Isolate cells from the obtained culture solution. Wash with water and dry
Parts of water and 97 parts of water are mixed to prepare a suspension cell suspension, and methacrylonitrile is continuously added dropwise at a rate of 3 parts per hour while controlling the pH to 8.5 with potassium hydroxide under stirring at 30 ° C.
It was made to react with. After reacting for 4 hours, addition of methacrylonitrile was stopped, and stirring was continued for further 30 minutes to allow almost complete reaction of methacrylonitrile in the system. After completion of the reaction, cells were removed by centrifugation to obtain a clear liquid. When the methacrylamide of this product was quantified, the content was 13.0% by weight. The yield of methacrylamide was about 95%.

Example 2 (1) Culture AK32 was cultured under the following conditions.

1) Glucose 1.0% by weight Meat extract 1.0 〃 Peptone 1.0 〃 Isobutyronitrile (inducer) 0.25 〃 Salt 0.1 〃 Potassium phosphate 0.1 〃 Magnesium sulfate 0.05 〃 Ferrous sulfate 0.005 〃 Manganese sulfate 0.005 〃 Ammonium sulfate 0.1 〃 Potassium nitrate 0.1 〃 pH 7.0 2) Culture conditions 30 ° C / 3 days (2) Hydration reaction of nitrile The cells were obtained from the obtained culture broth by the same method as in Example 1.

The reaction conditions are as follows.

Dry cell mass 0.2% by weight Substrate nitrile 2.0 〃 0.05M phosphate buffer (pH 7.0) 97.8 〃 Reaction temperature 10 ℃ Reaction time 5 minutes The results obtained are shown in the following table.

The analysis was usually performed by gas chromatography,
The amidation products of succinonitrile, adiponitrile, benzonitrile, terephthalonitrile, and nicotinonitrile, which have high boiling points, were analyzed by liquid chromatography.

Example 3 (1) Culture AK33 was cultured under the same conditions as in Example 2.

(2) Hydration reaction of acrylonitrile Bacterial cells were obtained from the obtained culture solution in the same manner as in Example 1, and 10 g of the dry cell weight was added to 0.05 M phosphate buffer (pH 7.0) 1, A suspension cell suspension was prepared. Acrylonitrile was intermittently added to this solution to carry out its hydration reaction. That is, acrylonitrile every 20 minutes
20 g each was added and the reaction was carried out at 2-3 ° C. The concentrations of unreacted acrylonitrile and acrylamide produced in the reaction mixture were determined by analyzing the reaction mixture immediately before the addition of acrylonitrile by gas chromatography. The amount of acrylamide in the reaction mixture increased almost linearly after the start of the reaction, and 450 g / -buffer of acrylamide was produced 5 hours after the start of the reaction. The reaction still seemed to proceed, but at this point the reaction rate slowed slightly and was stopped. The acrylamide yield was almost 100%, and acrylic acid as a by-product was only about 0.1% with respect to the added acrylonitrile.

Comparative Example 2 (1) Cultured Pseudomonas Chloro
The raphis) B23 strain (Microtechnology Research Institute No. 187) was cultured under the following conditions.

1) Medium Dextrin 0.5 wt% Dibasic potassium phosphate 0.2〃 Magnesium sulfate 0.02〃 Isobutyronitrile (inducer) 0.2〃 Salt 0.1〃 pH 7.0 2) Culture conditions 28 ℃ / 3 days (2) Hydration reaction of acrylonitrile Cells were separated from the obtained culture broth, washed with water, and acrylonitrile was intermittently added to a resting cell suspension prepared by dispersing the cells in phosphate buffer (pH 7.0) to a concentration of 20 g / dry cell weight. Hydration reaction was carried out.
The reaction is carried out at 0-4 ° C and the concentration of acrylonitrile is 0.4M.
Acrylonitrile was added intermittently so that The amount of acrylamide in the reaction mixture increased almost linearly after the reaction started, and 400 g / acrylamide was produced 7.5 hours after the reaction started. The reaction still seemed to proceed sufficiently, but at this point the reaction became viscous and was stopped.

The yield of acrylamide was about 99% and acrylic acid as a by-product was about 0.7% of the added acrylonitrile.

Example 4 (1) Culture AK32 was cultured under the same conditions as in Example 2.

(2) Methacrylonitrile hydration reaction The bacterial cells were obtained in the same manner as in Example 3, and 5 g of the dry bacterial cell weight was added to 0.05 M phosphate buffer (pH 7.0) 1 to give resting bacterial cells. A dispersion was prepared. Methacrylonitrile was intermittently added to this solution to carry out its hydration reaction.
That is, every 1 hour, 20 g of methacrylonitrile was added and the reaction was carried out at 2-3 ° C. The concentration of unreacted methacrylonitrile in the reaction mixture was quantified by analyzing the reaction mixture immediately before the addition of methacrylonitrile by gas chromatography. Around 5 hours after the start of the reaction, methacrylamide began to precipitate as crystals. After that, some unreacted methacrylonitrile began to remain, so the addition of methacrylonitrile was changed to 20 g every 2 hours, and the reaction was continued for 25 hours. As a result, 245 g of methacrylamide crystals were obtained. After the reaction solution was refrigerated at 4 ° C for 1600 hours, 20 g of methacrylonitrile was added,
When the reaction was carried out at ° C, unreacted methacrylonitrile was not detected at all after 2 hours, and it was converted to methacrylamide at a yield of almost 100%. Methacrylic acid as a by-product was only about 0.1% of the added methacrylonitrile.

Example 5 (1) Culture AK32 was cultured under the same conditions as in Example 2.

(2) Hydration reaction of methacrylonitrile After obtaining the cells by the same method as in Example 3, immobilized cells were prepared using calcium alginate gel. Methacrylonitrile was intermittently added to a suspension-immobilized bacterial cell dispersion prepared by adding 25 g of immobilized bacterial cells (0.5 g of dry bacterial cell weight conversion) to 75 g of 0.5 wt% calcium chloride aqueous solution (pH 7.0). A hydration reaction was performed. That is, 2 g of methacrylonitrile was added every hour and the reaction was carried out at 2-3 ° C. The concentration of unreacted methacrylonitrile in the reaction mixture was quantified by analyzing the reaction mixture immediately before the addition of methacrylonitrile by gas chromatography. Around 5 hours after the start of the reaction, methacrylamide began to precipitate as crystals. After that, some unreacted methacrylonitrile began to remain, so the addition of methacrylonitrile was changed to 2 g every 2 hours, and the reaction was continued for 15 hours. As a result, 10.8 g of methacrylamide crystals were obtained. Methacrylic acid as a by-product was only about 0.3% of the added methacrylonitrile.

Reference Example (1) Culture The AK3132 was cultured under the following conditions.

1) Medium Isobutyronitrile (inducer) 0.5 wt% Potassium phosphate 0.1 〃 Magnesium sulfate 0.05 〃 Ferrous sulfate 0.005 〃 Manganese sulfate 0.005 〃 Ammonium sulfate 0.1 〃 Potassium nitrate 0.1 〃 pH 7.0 2) Culture conditions 30 ℃ / 5 days (2) Hydration reaction of methacrylonitrile The bacterial cells were obtained from the obtained culture solution in the same manner as in Example 1 and used for the reaction. That is, a reaction solution containing 1.0% by weight of dry cells, 1.0% by weight of methacrylonitrile, and 98.0% by weight of 0.05M phosphate buffer solution (pH 7.0) was prepared at 30 ° C.
The reaction started at. After 30 minutes from the start of the reaction, the reaction solution was analyzed by gas chromatography to find that it was 0.1% by weight.
The unreacted methacrylonitrile remained almost quantitatively except that methacrylamide was produced.

Example 6 200 g of the medium described in Example 2 was placed in each of two 500 ml Erlenmeyer flasks and AK33 was placed on a rotary shaker at 30 ° C.
It was cultured at 220 rpm for 24 hours. In one of the two flasks, the culture was suspended for 24 hours to measure the bacterial cell concentration, and the turbidity was measured. As a result, it was found that 1050 ppm of bacterial cells were growing. In the other flasks, methacrylonitrile was added in an amount of 0.25 g per hour, and the culture was further continued under the conditions described above. The culturing was stopped 72 hours after the start of culturing, and the bacterial concentration in the flask and the concentration of methacrylamide were measured.As a result, it was found that the bacterial cells had grown to 5100 ppm, and 11.2 g of methacrylamide was accumulated in the flask. It was The amount of methacrylamide was quantified by gas chromatography. At this time, methacrylic acid was not detected in the culture solution, but methacrylonitrile remained at 500 ppm.

Example 7 (1) Culture AK32 was cultured under the same conditions as in Example 2.

(2) Hydration reaction of methacrylonitrile 20 g of the culture solution containing the bacterial cells was ingested in a 50 ml Erlenmeyer flask containing a magnetic rotor, and further placed in a water bath set at 4 ° C. while stirring with a magnetic stirrer.
Left for hours. Then 0.4g of methacrylonitrile per hour
When added one by one, methacrylamide crystals began to precipitate from 8 hours after the start of addition. When the addition of methacrylonitrile was continued, the experiment was terminated because the rotor could not rotate due to the crystals precipitated in the flask after 18 hours. The total slurry in the flask was diluted with water to 1000 g, and after dissolving the methacrylamide crystals, the concentration of methacrylamide was analyzed by gas chromatography to find that it was 0.91% by weight.
At this time, methacrylonitrile and methacrylic acid were not detected.

Example 8 (1) Culture AK32 was cultured in the same manner as in Example 2.

(2) Hydration reaction of methacrylonitrile The cells were collected from the obtained culture broth by centrifugation, washed with an aqueous potassium chloride solution (0.05 M, pH 7.0), and then centrifuged again. 2 g of resting AK32 cells (water content 80%) and 0.05 g phosphate buffer (pH 8.0) 15 g
40 g of glass beads having a diameter of 0.17 to 1.18 mm was charged into a homogenizer, and the cells were homogenized for 1.5 minutes while being blown with CO ₂ and cooled to 0 to 5 ° C. to crush the microbial cells. 3 g of this crushed product was taken out, and a reaction solution containing 98.0% by weight of the crushed product and 2.0% by weight of methacrylonitrile was prepared, and the same as in Example 1
The hydration reaction of methacrylonitrile was started. When the reaction solution was analyzed 5 minutes after the start of the reaction, formation of 2.5% by weight of methacrylamide was observed, and the yield of methacrylamide was about 100%.

Continuation of the front page (56) References JP-A-61-162193 (JP, A)

Claims (6)

[Claims] 1. A method for producing an amide, wherein a raw material nitrile is subjected to a hydration reaction by the action of a microorganism to be converted into a corresponding amide, and the resulting amide is separated from a reaction mixture.
As the microorganism, Rhodococcus sp. AK32 strain (Rhodococcus
sp.AK32) (Mikori Kenjoyori No. 1046) and Rhodococcus s
p.AK33 strain (Rhodococcus sp.AK33)
No. 7) using a strain selected from the group consisting of, and for culturing the strain, a derivatized nitrile selected from the group consisting of propionitrile, isobutyronitrile and methacrylonitrile. Production method. 2. The raw material nitrile is one selected from an aliphatic saturated nitrile, an aliphatic unsaturated nitrile, an aromatic nitrile and a heterocyclic nitrile, and in the claim (1), The method described. 3. The aliphatic saturated nitrile is acetonitrile,
The method according to claim (2), wherein the method is one selected from propionitrile, succinonitrile, and adiponitrile. 4. The method according to claim 2, wherein the aliphatic unsaturated nitrile is acrylonitrile or methacrylonitrile. 5. The method according to claim 2, wherein the aromatic nitrile is benzonitrile or phthalodinitrile. 6. The method according to claim (2), wherein the heterocyclic nitrile is nicotinonitrile.